Lens is an avascular tissue and is coupled electrically and metabolically by a network of communicating gap junctions. Although a number of intrinsic proteins have been identified in the lens membranes, majority are localized in the outer cortical fibers only. Ubiquitous presence of MIP26 in all regions of the lens fiber cell plasma membrane supports the view that it may be involved in gap junctional activities, and in maintaining structural integrity of the membrane. Aging and diabetes are the major factors promoting lens opacification. Diabetics are four to six times more likely to develop cataracts at an younger age than normal population. Elevated plasma glucose and resultant non-enzymatic glycosylation (glycation) of, proteins has been implicated in diabetic complications including cataract. Lysine and the free N-terminus are the most favorable sites for glycation. All the lysines (a total of three) are located on C-terminus arm of MIP26, and this arm has been proposed to be the channel gate; it also appears to have a binding site for calmodulin. Calmodulin, in the presence of Ca++, has been shown to regulate the channel uncoupling in reconstituted liposomes. Glycation of lysines may bring about a decrease in the net positive charge influencing the channel properties of MIP26. The long-term objective of this project is to understand the role of gap junctional channels in the development of cataract and to see how glycation of MIP26 influences the membrane function. This project has four major goals: (1) To identify the in vivo sites of glycation in streptozotocin-induced diabetic rat lens MIP26 (HPLC purified) using peptide mapping, amino acid analysis and microsequencing, and to show a relationship, if any, between glycation of specific sites to the progression of lens opacification as evaluated by Scheimpflug photography and densitometry. (2) To determine the effect of glycation of MIP26 on permeability and calmodulin-mediated channel gating in reconstituted liposomes. (3) To identify in vitro sites of glycation in calf lens MIP26 (HPLC purified) and to show a relationship, if any, between glycation of specific sites and altered permeability and gating properties. (4) To elucidate the role of each lysine on the gap junctional activity by generating mutated proteins where the basic lysines substituted with neutral asparagines using site-directed mutagenesis. These studies should lead to improved understanding of post-translational protein modifications of the putative gap junctional protein, MIP26, and its role in cataractogenesis.